Production of fermentation product

ABSTRACT

A process of producing fermentation product comprising the steps of, (i) forming an acidified suspension of particulate plant derived material comprising a first polysaccharide which is more readily hydrolysable and a second polysaccharide which is more difficult to hydrolysable, (ii) allowing the first polysaccharide to undergo hydrolysis by action of the acid at a temperature of at least 50° C. under conditions such that the first polysaccharide is hydrolysed and thereby forming a mixture of an aqueous liquor containing dissolved sugar and a solid residue containing the second polysaccharide, (iii) subjecting the mixture to one or more separation stages in which the solid residue and aqueous sugar liquor are substantially separated from each other, (iv) optionally washing the residue substantially free of acid and sugar, (v) adjusting the pH of the aqueous liquor to at least 4, (vi) passing the aqueous liquor from step (iv) into a fermentation stage where the dissolved sugars arc acted upon by a microorganism in a fermentation broth to produce a fermentation product, (vii) contacting the second polysaccharide by an enzyme, said enzyme hydrolyses the second polysaccharide to the component sugars, and allowing the component sugars to be acted upon by a microorganism in the fermentation broth to produce the fermentation product, (viii) separating the fermentation product from the broth, characterised in that the separation stage(s) in step (iii) is/are assisted by flocculation of the solid by-product, employing one or more flocculating agent(s) selected from the group consisting of water soluble polymers, water swellable polymers and charged microparticulate material. Typically such fermentation products include for instance ethanol, glycerol, acetone, n-butanol, butanediol, isopropanol, butyric acid, methane, citric Acid, fumaric acid, lactic acid, propionic acid, succinic acid, itaconic acid, acetic acid, acetaldehyde, 3-hydroxypropionic acid, glyconic acid, tartaric acid and amino acids such as L-glutaric acid, L-lysine, L-aspartic add, L-tryptophan, L-arylglycines or salts of any of these acids.

The present invention relates to processes of treating plant derivedmaterial to provide an aqueous liquor containing sugars which are usedin a fermentation process to produce a fermentation product. Typicallysuch fermentation products include for instance ethanol, glycerol,acetone, n-butanol, butanediol, isopropanol, butyric acid, methane,citric acid, fumaric acid, lactic acid, propionic acid, succinic acid,itaconic acid, acetic acid, acetaldehyde, 3-hydroxypropionic acid,glyconic acid, tartaric acid and amino acids such as L-glutaric acid,L-lysine, L-aspartic acid, L-tryptophan, L-arylglycines or salts of anyof these acids. It is known to treat a biomass with acid in order tohydrolyse polysaccharides to the component sugars that can be used in afermentation process to produce a fermentation product. For instanceU.S. Pat. No. 4384897 describes a method of treating biomass material inwhich it is subjected to a two stage hydrolysis in which polysaccharidesthat are more easily hydrolysed, such as hemicellulose and then in asecond stage the more difficult to depolymerise material e.g. cellulose,is depolymerised using a more severe hydrolytic treatment. The productsof the first and second stages include sugar solutions, organic acidsand aldehydes. The monosaccharides are subjected to fermentation toproduce ethanol and the beer resulting from the fermentation may then besubjected to rectification to produce ethanol of commercial grade. U.S.Pat. No. 4384897 sets out to provide improvements in the more efficientwashing of solids, the use of co-current washing or countercurrentwashing of solids and proposes the use of ferric and or aluminium ionsas flocculating agents to separate finely dispersed solids resultingfrom the neutralisation of the hydrolysate liquor stream.

Kyoung Heon Kim et al (Applied Biochemistry and Biotechnology, Vol91-93, pg 253-267) investigates the continuous countercurrent hydrolysisand extraction of hemicellulose from acid pretreated wood residues andconsiders the effect on drainage rate of such a pretreated biomass. Acontinuous countercurrent screw extractor used relies on the percolationof water by gravity through the pretreated biomass. One difficultyidentified is that the pretreated biomass has poor water drainageproperties and channelling or blockage may occur inside the extractor,which can result in low sugar recovery or low throughput.

It would be desirable to improve the drainage properties of acid treatedplant derived material in order to maximise sugar recovery.

It is also known from aNational Renewable Energy Laboratory (NREL)report entitled “Lignocellulose Biomass to Ethanol Process Design andEconomics of Co-Current Dilute Acid Prehydrolysis and EnzymaticHydrolysis Current and Future Scenarios” NREL/IP-580-26157 (July 1999)to treat cellulose as the second polysaccharide by a cellulase enzyme inorder to hydrolyse the cellulose into its component sugars. In one formof this process the solid by-product residue resulting from the firsthydrolysis step and containing cellulose is divided into a main streamand a secondary stream. The main stream is fed directly into thefermentation vessel and the secondary stream is passed to a cellulaseproduction stage, in which fungi are allowed to grow and act upon thecellulose, such that sugars and cellulase are formed. The sugars andcellulase are then fed into the fermentation vessel and the cellulaseacts upon the cellulose from the main stream and converts it into thecomponent sugars that in turn can be fermented to generate thefermentation product.

It is normally necessary to wash the solid by-product in order to ensurethat it is substantially free of acid and in particular acetic acidwhich is used during the hydrolysis of hemicellulose. It is necessary todo this since the acetic acid or other impurities could poison the fungiused in the production of cellulase or the cellulase produced therefrom.Normally the wash water is recycled water, for instance water that hasbeen separated from the still bottoms liquor in the distillationrecovery of the fermentation product in which suspended solids have beenremoved.

Since the wash water may contain other impurities that could be harmfulto either the cellulase, or fermentation processes it would be desirableto minimise the amount of wash water used.

A particular problem that can occur is that the efficiency of theprocess can be variable and sometimes resulting in diminished productionof the fermentation product. There is a need to further improve theyield of fermentation product produced in this process and to increasethe production rate.

According to the present invention we provide a process of producingfermentation product comprising the steps of,

-   -   (i) forming an acidified suspension of particulate plant derived        material comprising a first polysaccharide which is more readily        hydrolysable and a second polysaccharide which is more difficult        to hydrolyse,    -   (ii) allowing the first polysaccharide to undergo hydrolysis by        action of the acid at a temperature of at least 50° C. under        conditions such that the first polysaccharide is hydrolysed and        thereby forming a mixture of an aqueous liquor containing        dissolved sugar and a solid residue containing the second        polysaccharide,    -   (iii) subjecting the mixture to one or more separation stages in        which the solid residue and aqueous sugar liquor are        substantially separated from each other,    -   (iv) optionally washing the residue substantially free of acid        and sugar,    -   (v) adjusting the pH of the aqueous liquor to at least 4,    -   (vi) passing the aqueous liquor from step (iv) into a        fermentation stage where the dissolved sugars are acted upon by        a microorganism in a fermentation broth to produce a        fermentation product,    -   (vii) contacting the second polysaccharide by an enzyme, said        enzyme hydrolyses the second polysaccharide to the component        sugars, and allowing the component sugars to be acted upon by a        microorganism in the fermentation broth to produce the        fermentation product,    -   (viii) separating the fermentation product from the broth,        characterised in that the separation stage(s) in step (iii)        is/are assisted by flocculation of the solid by-product,        employing one or more flocculating agent(s) selected from the        group consisting of water-soluble or water-swellable natural,        semi-natural and synthetic polymers and charged microparticulate        materials.

We have found that surprisingly by using the special flocculationprocess in the separation stage, a consistently high yield offermentation product can be achieved. It is thought that the residualacid and sugar in the solid residue feed interferes with the formationof the enzyme and the action of the enzyme on the second polysaccharidein the solid residue. This in turn results in incomplete conversion ofthe second polysaccharide into the component sugars, which in turnresults in a reduced yield of fermentation product.

The improved separation stage in the process also has the advantage thatthe sugar solution resulting from the first hydrolysis stage issubstantially free from extraneous solid material, such as cellulosicfibres.

According to the process of the present invention, the enzyme which actson the second polysaccharide may be introduced directly into the solidby-product residue once it has been separated from the sugar liquorresulting from the hydrolysis of the first polysaccharide. or it may beadded once the second polysaccharide has been introduced into thefermentation process. The enzyme may be introduced by allowing a fungusto grow on the second polysaccharide, in which the fungus generates anenzyme which hydrolyses the polysaccharide into its component sugars.

The fungus capable of producing suitable enzymes may be Trichodermareesei, Aspergillus niger, Humicola insolens and Thermnomonospora fusca.

Alternatively the solid by-product containing the second polysaccharideis fed into the fermentation vessel and a commercially available enzymeis added directly into the fermentation vessel in order to act upon thesecond polysaccharide.

In one preferred form of the invention the solid residue of step (iv)comprising the second polysaccharide is divided into a main stream and asecondary stream. The main stream is passed directly into thefermentation stage, but the secondary stream of polysaccharide residueis passed into an enzyme production stage. In this stage the enzyme isgenerated by allowing fungi to act on the polysaccharide residue, andthis results in the formation of enzyme and converts the secondpolysaccharide into its component sugars. The enzyme and resultingsugars are passed into the fermentation vessel. The enzyme processresults in the production of sufficient enzyme to act upon the secondpolysaccharide introduced into the fermentation stage from the mainstream. Therefore the second polysaccharide in the fermentation vesselis then hydrolysed to the component sugars.

Alternatively all of the solid residue of step (iv) comprising thesecond polysaccharide is passed to an enzyme treatment stage in whichenzyme is generated by allowing fungi to grow on the polysaccharide. Theenzyme hydrolyses the polysaccharide into the component sugars and thenpassing the sugars into the fermentation stage in which the sugars areconverted into the fermentation product. However, the increasingconcentration of sugars could inhibit the process of enzyme productionand so it may be necessary to continually remove sugars that are formed.

The plant derived material is typically any readily available source ofpolysaccharides, particularly cellulosic materials. Typically thecellulosic material comprises materials selected from the groupconsisting of herbaceous biomass, softwood biomass, hardwood biomass,sewage sludge, paper mill sludge and the biomass fraction of municipalsolid waste. The herbaceous biomass may for instance be bagasse, ricestraw, rice hulls corn stover, wheat straw, grass, trees and cotton gintrash.

Preferably the plant derived material is cellulosic and compriseshemicellulose as the first polysaccharide and cellulose as the secondpolysaccharide. Generally the plant derived material also containslignin or lignin type materials, which remain in the solid by-product.

The acidified suspension may be formed by combining a particulatematerial comprising cellulose, hemicellulose and lignin with a diluteacid. Alternatively the suspension can be made by treatment of acellulosic biomass with sulphur dioxide gas, steam and water at anelevated temperature. Typically the process can be conducted byimpregnation of the biomass material with SO₂ gas followed by steam at205 to 215° C. for 5 minutes and then the addition of water to form aslurry (Stenberg et al., Recycling of Process Streams, AppliedBiochemistry, Vol 70-72, 1998, page 697-707, 1998).

By dilute we mean that the acid generally has a concentration of lessthan 10% by weight. Usually though the concentration will be much lower,for instance less than 5%. The acid may be a strong mineral acid such ashydrochloric acid, sulphuric acid, sulphurous acid, hydrofluoric acid,nitric acid and phosphoric acid. Alternatively the acid may be anorganic acid, for example carbonic acid, tartaric acid, glucuronic acid,formic acid, trichloroacetic acid or other carboxylic acids.

The acid ideally exhibits a pKa below 4. Preferred results are obtainedby using either hydrochloric acid or sulphuric acid.

The hydrolysis of the first polysaccharide is preferably carried out ata temperature of between 120 and 220° C. for a period of 1 to 15minutes, although lower temperatures are possible if the treatment timeis longer.

In each of the first and second hydrolysis stages, the resultinghydrolysate is then separated from the solid materials, preferablythrough pressing of the treated material to separate the residue as asolid product. The solid product that is separated may be subjected toat least one wash cycle to remove any residual sugar solution from thesolid. The wash cycle comprises washing the solid product with asuitable wash liquid. The wash liquid may be water. Normally the washwater is recycled water, for instance water that has been separated fromthe still bottoms liquor in the distillation recovery of thefermentation product in which suspended solids have been removed.

The liquid hydrolysate which contains sugars and acid can then becollected for further processing. When the first polysaccharide ishemicellulose, the resulting hydrolysate is generally C₅ sugars and whenthe second polysaccharide is cellulose the hydrolysate is generally C₆sugars.

In each case it is important to adjust the pH of the acid sugar liquorsto a pH value of at least 4. The pH adjustment may be done by additionof a base or by use of an ion exchange resin, which is capable ofneutralising the acid. Preferably the pH of the acidified aqueous sugarliquor that results from the digestion process is adjusted to a pH of atleast 10 by addition of a basic material such as sodium carbonate, andthen subsequent adjustment of the pH to a more neutral or slightlyacidic pH. Desirably the pH may be adjusted to a value of between 10 and12, preferably about 11, by addition of a base, followed by titrating topH 4 and 5, preferably about pH 4.5.

Alternatively the acid may be removed from the liquor by passing thehydrolysate through a bed of resin beads to remove the acid. The aqueoussugar stream that desirably contains at least 98% of the sugar presentin the hydrolysate can then be recovered.

After the separation of the acid from the sugar stream, the acid ispreferably concentrated for reusefor example by evaporation.

The fermentation process of the present invention typically involvesallowing the fermentation to proceed for 3 to 5 days. Volatilefermentation products may be continually removed by recirculating carbondioxide through a cooled condensing column. Desirably the fermentationproducts are collected from the condensing column after three to fivedays and then distilled. Preferably volatile fermentation products areseparated from the broth by passing the broth comprising thefermentation product into a distillation stage, where the fermentationcompound is collected as a distillate and the residue ‘still bottoms’ isremoved. Microorganisms can be separated from the fermentation broth orpreferably from the still bottoms, preferably through centrifugation andcan be recycled for reuse. In one preferred aspect of the invention thefermentation product is separated from the broth by passing the brothcomprising the fermentation product into a concentration stage, in whichthe fermentation compound is collected in the concentrate and extractedby at least one means selected from the group consisting of ionexchange, solvent extraction and electrodialysis.

The process can be used to prepare a range of fermentation products, butpreferably the fermentation product is selected from the groupconsisting of ethanol, glycerol, acetone, n-butanol, butanediol,isopropanol, butyric acid, methane, citric acid, fumaric acid, lacticacid, propionic acid, succinic acid, itaconic acid, acetic acid,acetaldehyde, 3-hydroxypropionic acid, glyconic acid, tartaric acid andamino acids such as L-glutaric acid, L-lysine, L-aspartic acid,L-tryptophan, L-arylglycines or salts of any of these acids.

The microorganisms used in the fermentation process of the presentinvention can be, for example, a yeast such as Klyveromyces species,Candida species, Pichia species, Brettanomyces species, Saccharomycesspecies such as Saccharomyces cerevisiae and Saccharomyces uvarum,Hansenula species and Pachysolen species. Alternatively, themicroorganism can be a bacterial species such as Leuconostoc,Enterobacter, Klebsiella, Erwinia, Serratia, Lactobacillus, Lactococcus,Pediococcus, Clostridium, Acetobacter, Gluconobacter, Lactobacillus,Aspergillus, Propionibactedum, Rhizopus and Zymomonas mobils. Inaddition genetically modified strains may also be used.

Since the solid product generally comprises lignin and analogousmaterials it can be particularly difficult to separate from the liquor.We have unexpectedly found that the production of fermentation productcan be significantly improved by applying one or more flocculatingsagent to the separation of the hydrolysate from the solid product. Wehave found that the solid product can be more efficiently dewatered bythe process and that a higher cake solids can be achieved. Since thesolid product can be more efficiently dewatered there is a reducedrequirement for separation equipment capacity and equipment that is lesscapital intensive and less expensive to operate, such as a filter press,can be used. Since higher cake solids can be achieved, less of the acidsugar solution remains in the residual by-product solid. Hence thequantity of water required to wash the by-product solid free of acid andsugar is much reduced, improving both the productivity and efficiency ofthe process.

Suitably the flocculating agent is selected from the group consisting ofwater soluble or water swellable natural, semi-natural and syntheticpolymers. Preferably the polymer is synthetic and may be formed bypolymerisation of at least one cationic, non-ionic or and/or anionicmonomer(s) alone or with other water soluble monomers. By water solublewe mean that the monomer has a solubility of at least 5 g/100 ml at 25°C.

Preferably polymeric flocculating agents are formed from ethylenicallyunsaturated water soluble monomers that readily polymerise to producehigh molecular weight polymers. Particularly preferred polymers includemonomers that are selected from the group consisting of polyacrylatesalts, polyacrylamide, copolymers of acrylamide with (meth) acrylic acidor salts thereof, copolymers of acrylamide with dialkylaminoalkyl (meth)acrylate or acid addition or quatemary ammonium salts, polymers ofdiallyidimethyl ammonium chloride, polyamines and polyethylene imines.The polymers may be linear, branched or cross-linked.

The polymers may be prepared by any convenient conventional process, forinstance by solution polymerisation, gel polymerisation, reverse phasesuspension polymerisation and reverse phase emulsion polymerisation.Suitable processes include those described in EP-A-150933 orEP-A-102759.

Suitable polymers are anionic, cationic and non-ionic polymers. Thepreferred polymers are non-ionic and cationic polymers of sufficientlyhigh molecular weight such that it exhibits an intrinsic viscosity of atleast 4 dl/g. Such an intrinsic viscosity generally indicates a polymerof several million molecular weight, for instance generally greater than5,000,000 and usually at least 7,000,000. In general the polymerpreferably has an intrinsic viscosity greater than 6 dl/g, often atleast 8 or 9 dl/g. The intrinsic viscosity can be as high as 30 dl/g orhigher. In many cases though suitable cationic polymers exhibit anintrinsic viscosity in the range of 7 to 25 dl/g, in particular 10 to 20dl/g, in particular around 14 or 15 dl/g.

Suitable cationic monomers include quaternary ammonium or acid salts ofmonomers which contain amine groups. Preferably the cationic polymer isformed from a monomer or blend of monomers comprising at least onecationic monomer selected from the group consisting of quaternaryammonium and acid salts of dimethylaminoethyl (meth) acrylate,quaternary ammonium and acid salts of dimethylaminoethyl (meth)acrylamide and diallyldimethyl ammonium chloride. The cationic monomersmay be hompolymerised or copolymerised with other monomers, for instanceacrylamide. The cationic polymers thus may be any polymer that carries acationic, provided of course that they are of sufficiently highmolecular weight to exhibit an intrinsic viscosity of at least 4 dl/g.Intrinsic viscosity is measured using a suspended level viscometer in 1M NaCl buffered to pH 7.5 at 25° C.

The cationic polymers according to the invention may be prepared assubstantially linear polymers or as branched or structured polymers.Structured or branched polymers are usually prepared by inclusion ofpolyethylenically unsaturated monomers, such as methylene-bis-acrylamideinto the monomer mix, for instance as given in EP-B-202780. Preferablyhowever, the polymers are substantially linear and are prepared in theform of a bead or powdered product.

Suitably the polymeric flocculating agent would be added as an aqueoussolution or aqueous dispersion. The polymer may be added in an amountsufficient to effect flocculation. Typically the amount of polymericflocculating agent sufficient to induce flocculation would be usually atleast 0.002 weight % based on weight of suspended solids. Usually betterflocculation and therefore separation can be achieved if at least 0.01%is used. The dose may be substantially higher, for instance up to 1%.However, optimum flocculation and separation is normally achieved usingdoses in the range of 0.015% to 0.2%, especially 0.02% to 0.1%.Following flocculation of the suspended solids the solid product can beseparated from the hydrolysate aqueous liquor by mechanical means, forinstance filter press, centrifuge, belt press, horizontal belt filter orpressure filter. The action of the flocculating agent greatly enhancesthe separation of the solids from the liquor by comparison to separationusing solely mechanical means. We have found that the process of thepresent invention provides a higher cake solids, with less trappedresidual aqueous liquor, which means that a higher proportion of thesugar liquor is available for conversion into the fermentation product.Likewise we find that the aqueous liquor contains much lower levels ofextraneous suspended cellulosic solids. Furthermore we also find thatless wash water is required.

The solid product that results from the separation step should be as dryas possible in order to prevent any loss of sugar, which would otherwisebe used in the fermentation process.

In a further preferred embodiment of the present invention theflocculating agent is a charged microparticulate material. Particularlysuitable examples of charged microparticulate materials includeswellable clays, anionic, cationic or amphoteric microparticulate silicabased materials and organic cross-linked polymeric microparticles.

The siliceous material may be any of the materials selected from thegroup consisting of silica based particles, silica microgels, colloidalsilica, silica sols, silica gels, polysilicates, aluminosilicates,polyaluminosilicates, borosilicates, polyborosilicates, zeolites orswellable clay.

This siliceous material may be in the form of an anionicmicroparticulate material. Alternatively the siliceous material may be acationic silica. Desirably the siliceous material may be selected fromsilicas and polysilicates.

The polysilicates of the invention may be prepared by reducing the pH ofan aqueous solution of an alkali metal silicate. For instancepolysilicic microgels otherwise known as active silica may be preparedby acidification of alkali metal silicate to between pH 2 and 10 by useof mineral acids or acid exchange resins, acid salts and acid gases. Itmay be desired to age the freshly formed polysilicic acid in order toallow sufficient three dimensional network structure to form.

Generally the time of ageing is insufficient for the polysilicic acid togel. Particularly preferred siliceous material includepolyalumino-silicates. The polyaluminosilicates may be for instancealuminated polysilicic acid, made by first forming polysilicic acidmicroparticles and then post treating with aluminium salts.

Alternatively the polyaluminosilicates may be polyparticulatepolysicilic microgels of surface area in excess of 1000m²/g formed byreacting an alkali metal silicate with acid and water soluble aluminiumsalts. Typically the polyaluminosilicates may have a mole ratio ofalumina:silica of between 1:10 and 1:1500.

Polyaluminosilicates may be formed by reducing the pH of an aqueoussolution of alkali metal silicate to between pH 2 and 10 usingconcentrated sulphuric acid containing 0.2 to 2.0% by weight of a watersoluble aluminium salt, for instance aluminium sulphate. The aqueoussolution may be aged sufficiently for the three dimensional microgel toform. Typically the polyaluminosilicate is aged for up to about two anda half hours before diluting the aqueous polysilicate to 0.5 weight % ofsilica.

The siliceous material may be a colloidal borosilicate. The colloidalborosilicate may be prepared by contacting a dilute aqueous solution ofan alkali metal silicate with a cation exchange resin to produce asilicic acid and then forming a heel by mixing together a dilute aqueoussolution of an alkali metal borate with an alkali metal hydroxide toform an aqueous solution containing 0.01 to 30% B₂0₃, having a pH offrom 7 to 10.5.

The swellable clays may for instance be typically a bentonite type clay.The preferred clays are swellable in water and include clays which arenaturally water swellable or clays which can be modified, for instanceby ion exchange to render them water swellable. Suitable water swellableclays include but are not limited to clays often referred to ashectorite, smectites, montmorillonites, nontronites, saponite,sauconite, hormites, attapulgites and sepiolites.

Most preferably the clay is a bentonite type clay. The bentonite may beprovided as an alkali metal bentonite. Bentonites occur naturally eitheras alkaline bentonites, such as sodium bentonite or as the alkalineearth metal salt, usually the calcium or magnesium salt. Generally thealkaline earth metal bentonites are activated by treatment with sodiumcarbonate or sodium bicarbonate. Activated swellable bentonite clay isoften supplied as a dry powder. Alternatively the bentonite may beprovided as a high solids flowable slurry, for example at least 15 or20% solids.

When the charged microparticulate material comprises an organiccross-linked polymeric microparticles. The microparticles may be made asmicroemulsions by a process employing an aqueous solution comprising acationic or anionic monomer and crosslinking agent; an oil comprising asaturated hydrocarbon; and an effective amount of a surfactantsufficient to produce particles of less than about 0.75 micron inunswollen number average particle size diameter.

Microbeads are also made as microgels by procedures described by YingHuang et. al., Makromol. Chem. 186, 273-281 (1985) or may be obtainedcommercially as microlatices. The term “microparticle”, as used herein,is meant to include all of these configurations, i.e. beads per se,microgels and microlatices.

The charged microparticle material may be used in amounts of at least0.002% based on weight of suspended solids. Typically though the dosesare usually as high as 0.8 or 1.0% or higher. When the chargedmicroparticle material is inorganic, the dose is usually in excess of0.06%, preferably in the range 0.1 to 0.6%. When the chargedmicroparticle is organic the dose is typically below 0.3%, preferably inthe range 0.02 to 0.1%.

Unexpectedly we have found that the hydrolysate liquor can be separatedparticularly rapidly when the flocculation is effected by employing awater-soluble or water-swellable polymer and a charged microparticulatematerial. In one aspect we find that particularly effective flocculationand separation of the solids from the liquor is achieved whenflocculation is carried out by introducing an anionic microparticlematerial into the mixture and then reflocculating by adding a cationicor substantially non-ionic polymer. In a further preferred embodiment ofthe present invention we find that especially fast and efficientseparation of solids is achieved by a process in which flocculation iseffected by introducing a cationic polymer into the mixture and thenreflocculating by adding an anionic microparticulate material.

The following examples illustrate the invention.

EXAMPLE 1

Pre-hydrolysis: Milled wood chips steamed with low pressure steam toapproximately 100C. After steaming concentrated sulphuric acid isdiluted and added to the mixture until the mixture contains 0.52% acidand the solids in the reactor are 22% by weight. The mixture is thensteam heated to 175° C. for 15 minutes. The mixture is then flash cooledfor 15 minutes to remove 6.5% of the acetic acid and 61% of the furfuraland hydroxymethyl furfural.

Separation: The 26% insoluble solids present in the pre-hydrolysedslurry (containing 0.38% sulphuric acid) is then separated on a filterpress. Prior to pressing solutions of flocculant or flocculants (at 0.2to 0.5% solids) and/or particulate suspensions (at 0.2 to 0.5% solids)are added into the feed stream with necessary agitation at a dose of0.22 to 2 Kg per tonne of solids. Flocculants increase the rate of freedrainage by gravity through a porous belt, before preparation of afilter cake in a wedge zone and subsequent further dewatering in apressure zone. A method of reducing the toxins remaining in the liquidportion is to wash with (recycled) water.

After ion exchange for the removal of acetic acid, the liquid portion ofthe hydrolysate is acidified to pH 2 by the addition of sulphuric acid.Lime is then added to raise the pH to 10 and the liquor is then heatedto 50° C. The liquid is then adjusted to the fermentation pH of 4.5 for4 hours allowing gypsum crystals to form for separation by filtration.

Simultaneous Saccharification and Co-Fermentation (SSCF): Detoxified anddiluted hydrolysed solids is split to cellulase fermentations, Z.mobilis seed production and SSCF fermenters. The hydrolysate feed streamis 22% combined soluble and insoluble solids. The portion of hydrolysedsolid residue is that is split off for Z. mobilis seed production isapproximately 10%. The portion of hydrolysate split off for cellulaseproduction is dependent on both the cellulase yield on the xylose andcellulose present and the required loading of enzyme in the SSCF. Forcellulase production pre-hydrolysed solids-conditioned hydrolysateliquor, recycled water, corn steep liquor (to 1%) and nutrients((NH₄)₂SO₄, KH₂PO₄, MgSO₄.7H₂O CaCl₂.2H₂O and Tween 80) and corn oil asan antifoam (0.1% v/v) are combined to give a final celluloseconcentration of 4%. The batch is then run for 160 hours at 28° C. toproduce cellulase. For SSCF, detoxified hydrolysate slurry (22% totalsolids) is cooled to 30° C. and added to the fermenter together with a10%(v/v) seed inoculum. Corn steep liquor is added to 0.25% andcellulase to give a final concentration of 15 FPU/g cellulose and aninitial cellulose concentration of 22%. The SSCF fermentation in whichcellulose is converted to fermentable sugars by cellulase and thefermentable sugars converted to ethanol by Z. mobilis takes 7 days.

EXAMPLE 2

Pre-hydrolysis: Softwood chips (2 mm) with a dry solids content of 48%were added to 400 g of water and heated to 190° C. Once at 190° C.sulphuric acid was added to a concentration of 0.7% under nitrogenpressure and the mixture was left for 3 minutes. The temperature wasrapidly reduced to 80° C. and the insoluble solids present in thepre-hydrolysed slurry (containing 0.32% sulphuric acid) wass thenseparated on a filter press. Prior to pressing, solutions of flocculantor flocculants (at 0.2 to 0.5% solids) and/or particulate suspensions(at 0.5 to 15% solids) are injected into the slurry with necessaryagitation at a dose of 0.2 to 2 Kg per tonne of solids.

Enzymic hydrolysis: Tap water is added to the recovered pre-hydrolysesdsolid matter to adjust the dry matter content of the suspension to 7.5%(w/w). The pH was adjusted to 4.8 with calcium hydroxide and then 10%(w/w) sodium hydroxide was used to maintain the pH at 4.8 duringhydrolysis. To perform the hydrolysis cellulase (activity 75 FPU/g), wasadded at a dose of 0.175g/g of fibrous material supplemented with 0.025g/g cellobiase (β-galactosidase activity 400 IU/g). Hydrolysis wasallowed to proceed for 4 days. The solid residues were then separated ina filter press. Prior to pressing solutions of flocculant or flocculants(at 0.2 to 0.5% solids) or particulate suspensions (at 0.5 to 15%solids) are added into the slurry with necessary agitation at a dose of2 to 10 Kg per tonne of solids.

Fermentation: The hydrolysate was supplemented to a final concentrationof 0.5 g/L (NH₄)2SO₄ and 0.025 g/L MgSO₄.7H₂O and inoculated with yeastto a concentration of 1% (w/v). The fermentation was maintained at 30°C. and at pH 4.8 by the addition of 10% (w/w) sodium hydroxide.

EXAMPLE 3

The separation of acid and sugar from the fermantation product of theinvention was assessed using the equipment and the results obtained willnow be described by way of example in the accompanying drawings inwhich:

FIG. 1 is a diagrammatic axial section of a syringe,

FIG. 2 shows the syringe of FIG. 1 containing a sample to be tested,

FIG. 3 illustrates the introduction of flocculant into the sample,

FIG. 4 shows a test rig in part sectional side elevation,

FIG. 5 is a part sectional plan view of the rig of FIG. 4,

FIGS. 6 and 7 are vertical sections through a device for separatingliquid from the sample,

FIGS. 8 is a graph showing the cumulative conductance, which arises fromremoval of the acidin the separated liquid and

FIG. 9 is a graph of the cumulative amount of sugar removed in theseparated liquid.

Referring to FIG. 1 of the drawings an open ended syringe housing 10 ofcircular cross section is adapted to receive syringe plungers 12 and 14into each open end 16 and 18 respectively. As shown in FIG. 2 a sample20 of hydrolysate to be examined, optionally together with some ballbearings, is disposed in the syringe housing substantially in the midpart thereof and held in place by the plungers 12 and 14. The syringetogether with the sample is incubated for a period of time, for example15 minutes at a temperature which is typically about 90° C. Afterincubation one plunger is removed from the syringe and as shown in FIG.3 polymer flocculant 24 is introduced into the sample with a pipette 22.The removed plunger is replaced and the syringe shaken in order to tryto ensure that the polymer is distributed throughout the sample. Thesyringe is then incubated again for example for about ten minutes at atemperature of, for example, 90° C.

The speed at which liquid separates from the solids In the sample cannow be measured using the rig shown in FIGS. 4 and 5. This consists of avertically oriented tube 30 sized to receive the syringe at its upperend. The lower end of tube 30 is disposed just above a filter paper 32.Contacts 34 are provided adjacent the filter paper which are arranged tosupply a signal to a timer 36 to start the timer when liquid spreads tothe contacts 34 from the tube. A further contact 38 linked to the timeris arranged to turn the timer off when liquid from the tube reachescontact 38. Thus the rig measures the time taken for liquid to spreadacross the filter paper from contact 34 to contact 38. This is known asCapillary Suction Time (CST) and is measure of the speed of separationof liquid from solids in the test sample.

To obtain the CST for the sample one plunger is removed and the syringeis inserted into the tube 30, the other plunger being moved into thesyringe housing to bring the sample into contact with the filter paper32 as Illustrated in FIG. 4. Liquid separating from the sample spreadsacross the filter paper outwardly from the area of contact of the samplewith the filter paper starting the timer when it reaches contacts 34 andstopping the timer when it reaches contact 38.

Using the above described equipment the CST was determined for 5 g.samples of hydrolysate flocculant polymer being added as shown in thefollowing table: The target CST was 98.8. Polymer Addition CST secondsControl + 100 μl H₂O 127.9 100 μl Polymer 1  91.8

Polymer 1 is an acrylamide homopolymer with an IV of approx 15 dl/g.

EXAMPLE 4

Following the procedure described with reference to FIGS. 1 to 3 afterthe second incubation one of the plungers is removed from the syringeand as shown in FIGS. 6 and 7 the open end of the syringe inserted intothe open top of a larger syringe 40 having a perforated base 42 forsupporting a mesh 44. A receiving cylinder 46 is positioned around thelower end of syringe 40 and the assembly of syringe 40 and cylinder 46is mounted in flask 48 having a connection 50 to a vacuum. The sample iswashed and filtrate 52 collecting in the receiving cylinder can beexamined.

The equipment with reference to FIGS. 6 and 7 was used to examine theseparation of acid and sugar from a sample of hydrolysate treated inaccordance with invention.

A 5 g sample of hydrolysate derived from corn stover was placed in thesyringe housing 10 together with some ball bearings and held in placewith the plungers during Incubation. 0.1 ml of a 1% solution of Polymer1 was introduced into the sample by a pipette as illustrated in FIG. 2.After the second incubation the sample and flocculant was transferredfrom syringe housing 10 to syringe 40, a 58 micron mesh having beenprovided on the perforated base 42. 10 ml of wash water was delivered tothe syringe 40 while the vacuum was applied. The conductance of thefiltrate was measured for each 1 ml of liquid recovered and thecumulative conductance results are shown on the graph of FIG. 8 whichalso shows the results obtained from a control sample. As can be seenthe inclusion of the flocculant caused a rapid increase in theconductance on the addition of the wash water which indicates that acidis being removed.

FIG. 9 shows the increase in sugar concentration in the filtrate withthe addition of wash water.

EXAMPLE 5

Following the same procedure as in Example 4 the actual amount of sugarrecovered was evaluated with two different polymers at two differentpolymer concentrations. The tests were performed twice at eachconcentration and the results shown in the following Table 1. PolymerConcentration 200 ppm 600 ppm Control 190 mg 181 mg 249 mg 237 mg  7.8mls  7.4 mls  8.3 mls  7.8 mls Polymer 2 211 mg 200 mg 257 mg 244 mg 7.4 mls  7.0 mls  8.1 mls  7.7 mls Polymer 1 386 mg 367 mg 246 mg 233mg  7.2 mls  6.8 mls  8.9 mls  8.5 mls

Polymer 2 is a copolymer or 8% sodium acrylate 92% acrylamide, IV approx9 dl/g

The above results are based on recovery of about 95% of the wash water.Recalculating the figures on the theoretical basis that all 10 mls ofthe wash water is recovered the results are as follows: PolymerConcentration 200 ppm 600 ppm Control 243 mg 231 mg 300 mg 285 mgPolymer 2 285 mg 271 mg 317 mg 301 mg Polymer 1 536 mg 509 mg 276 mg 262mg

1. A process of producing fermentation product comprising the steps of, (i) forming an acidified suspension of particulate plant derived material comprising a first polysaccharide which is more readily hydrolysable and a second polysaccharide which is more difficult to hydrolyse, (ii) allowing the first polysaccharide to undergo hydrolysis by action of an acid at a temperature of at least 50° C. under conditions such that the first polysaccharide is hydrolysed and thereby forming a mixture of an aqueous liquor containing dissolved sugar and a solid residue containing the second polysaccharide, (iii) subjecting the mixture to one or more separation stages in which the solid residue and aqueous sugar liquor are substantially separated from each other, (iv) optionally washing the residue substantially free of acid and sugar, (v) adjusting the pH of the aqueous liquor to at least 4, (vi) passing the aqueous liquor from step (iv) into a fermentation stage where the dissolved sugars are acted upon by a microorganism in a fermentation broth to produce a fermentation product, (vii) contacting the second polysaccharide by an enzyme, said enzyme hydrolyses the second polysaccharide to the component sugars, and allowing the component sugars to be acted upon by a microorganism in the fermentation broth to produce the fermentation product, (viii) separating the fermentation product from the broth, characterised in that the separation stage(s) in step (iii) is/are assisted by flocculation of the solid by-product, employing one or more flocculating agent(s) selected from the group consisting of water-soluble polymers, water-swellable polymers and charged microparticulate material.
 2. A process according to claim 1 in which the solid residue of step (iv) comprising the second polysaccharide is divided into a main stream and a secondary stream, and passing the main stream directly into the fermentation stage, wherein the secondary stream of polysaccharide residue is passed into an enzyme production stage, in which enzyme is generated by allowing fungi to act on the polysaccharide residue, resulting in the formation of enzyme and sugars resulting from the second polysaccharide contained within the secondary stream, then passing the enzyme and sugars of step (vi) into the fermentation stage, wherein the enzyme acts on the second polysaccharide in the fermentation vessel and hydrolyses the second polysaccharide to the component sugars.
 3. A process according to claim 1 in which the solid residue of step (iv) comprising the second polysaccharide is passed to an enzyme treatment stage in which enzyme is generated by allowing fungi to grow on the polysaccharide and said enzyme hydrolyses the polysaccharide into the component sugars and then passing the resulting sugars into the fermentation stage in which the sugars are converted into the fermentation product.
 4. A process according to claim 1 in which the plant derived material comprises materials selected from the group consisting of herbaceous biomass, softwood biomass, hardwood biomass, sewage sludge, paper mill sludge and the biomass fraction of municipal solid waste.
 5. A process according to claim 1 in which the plant derived material is cellulosic and comprises hemicellulose as the first polysaccharide and cellulose as the second polysaccharide.
 6. A process according to claim 1 in which the acid has a pKa of below 4 and has a concentration up to 2% by weight.
 7. A process according to claim 1 in which the acid is selected from the group consisting of sulphuric acid and hydrochloric acid.
 8. A process according to claim 1 in which the hydrolysis of the first polysaccharide is conducted at a temperature of between 120 to 220° C. for a period of from 1 minute to 15 minutes.
 9. A process according to claim 1 in which the flocculating agent is selected from the group consisting of water soluble or water swellable natural, semi-natural and synthetic polymers.
 10. A process according to claim 9 in which the polymer is formed from a water soluble monomer or blend of monomers.
 11. A process according to claim 9 in which the polymer is selected from the group consisting of polyacrylate salts, polyacrylamide, copolymers of acrylamide with (meth) acrylic acid or salts thereof, copolymers of acrylamide with dialkylaminoalkyl (meth) acrylate or acid addition or quaternary ammonium salts, polymers of diallyldimethyl ammonium chloride, polyamines and polyethylene imines.
 12. A process according to claim 1 in which the flocculating agent is a charged microparticulate material.
 13. A process according to claim 12 in which the charged microparticulate material is selected from the group consisting of swellable clays, anionic, cationic or amphoteric microparticulate silica based materials and organic cross-linked polymeric microparticles.
 14. A process according to claim 1 in which flocculation is effected by employing a water soluble or water-swellable polymer and a charged microparticulate material.
 15. A process according to claim 1 in which flocculation is effected by introducing an anionic microparticle material into the mixture and then reflocculating by adding a substantially non-ionic polymer.
 16. A process according to claim 1 in which flocculation is effected by introducing a cationic polymer into the mixture and then reflocculating by adding an anionic microparticulate material.
 17. A process according to claim 1 in which flocculation is effected by introducing a cationic polymer into the mixture and then reflocculating by adding an anionic polymer.
 18. A process according to claim 1 in which flocculation is effected by introducing an anionic polymer into the mixture and then reflocculating by adding a cationic polymer.
 19. A process according to claim 1 in which the solid-by product material comprises lignin and analogous materials.
 20. A process according to claim 1 in which the fermentation product is selected from the group consisting of ethanol, glycerol, acetone, n-butanol, butanediol, isopropanol, butyric acid, methane, citric acid, fumaric acid, lactic acid, propionic acid, succinic acid, itaconic acid, acetic acid, acetaldehyde, 3-hydroxypropionic acid, glyconic acid, tartaric acid and amino acids wherein the amino acids are selected from the group consisting of L-glutaric acid, L-lysine, L-aspartic acid, L-tryptophan, L-arylglycines and salts of any of these acids.
 21. A process according to claim 1 in which the fermentation product is separated from the broth by passing the broth comprising the fermentation product into a distillation stage, where the fermentation compound is collected as a distillate and the residue ‘still bottoms’ is removed.
 22. A process according to claim 1 in which the fermentation product is separated from the broth by passing the broth comprising the fermentation product into a concentration stage, in which the fermentation compound is collected in the concentrate and extracted by at least one means selected from the group consisting of ion exchange, solvent extraction and electrodialysis. 